Elsevier

Brain Research

Volume 1326, 22 April 2010, Pages 162-173
Brain Research

Research Report
Regulation of spinal neuroimmune responses by prolonged morphine treatment in a rat model of cancer induced bone pain

https://doi.org/10.1016/j.brainres.2010.02.039Get rights and content

Abstract

Cancer induced bone pain (CIBP) is a major clinical problem. Although opioids remain the principal axis in drug therapies for CIBP, their sustained application is known to induce cellular and molecular adaptations including enhanced neuroimmune reactivity. This is generally characterized by glial activation and proinflammatory cytokine production which frequently results in pharmacological tolerance. This research was performed to investigate spinal neuroimmune responses after prolonged systemic morphine treatment in a rat model of CIBP. The model was established using a unilateral intra-tibia injection of Walker 256 mammary gland carcinoma cells. Subcutaneous morphine was repeatedly administered from postoperative days 14 to 19. Mechanical allodynia to von Frey filaments and ambulatory pain scores were recorded to investigate changes of nociceptive behaviors. Spinal glial activation was detected by immunohistochemistry and real-time PCR; the production of proinflammatory cytokines (IL-1β and TNF-α) was examined through real-time PCR and ELISA. Results showed that chronic morphine use failed to elicit analgesic tolerance in the rat CIBP model. Moreover, the treatment had no significant influence on the activated spinal glia morphology, cell density and expression of special cytomembrane markers, whereas it significantly down-regulated the local proinflammatory cytokine production at the mRNA and protein level. Collectively, these data suggest that chronic morphine treatment in CIBP is not concomitant with pharmacological tolerance, at least partially because the treatment fails to amplify spinal neuroimmune responses.

Introduction

Cancer induced bone pain (CIBP) remains a severe clinical issue, and it is a very complicated pain syndrome including localized ongoing background pain and spontaneous pain or movement-evoked (incident) pain (Mercadante and Arcuri, 1998, Portenoy and Frager, 1999, Urch, 2004). To date about 85% of patients with bone metastases suffer from this intolerable pathologic pain which is associated with increased morbidity, increased anxiety and depression, reduced performance status, and a reduced quality of life (Bruera and Kim, 2003). Although there have been several recent advances in the therapeutic management, the primary drugs remain mu opioid receptor analgesics so far, represented by such drugs as morphine (Cherny, 2000, Mercadante, 1999, Ruiz-Garcia and Lopez-Briz, 2008). The chronic nature of cancer pain often requires prolonged morphine application through controlled release tablets, repeated bolus injections, and so on (Clemens and Klaschik, 2007, Delaney et al., 2008, Quigley, 2005). Unfortunately, some evidence suggests that long-term morphine treatment may lead to cellar and molecular adaptations (Williams et al., 2001) that often result in pharmacological tolerance (i.e., a decreased analgesic effect with prolonged administration of a constant dose) (Bohn et al., 2000).

Most animal studies regarding opioid tolerance have only addressed neuronal mechanisms, and have ignored other potential modulators. Recent evidence has demonstrated that spinal neuroimmune responses, which are characterized by glial activation and proinflammatory cytokine production, make a great contribution to the generation and maintenance of opioid tolerance (Raghavendra et al., 2002, Raghavendra et al., 2004a, Tawfik et al., 2005). Since the first study revealed that spinal neuroimmune reactivity was closely related to morphine tolerance, evidence rapidly accrues that chronic morphine treatment can directly activate both astrocytes and microglia (Cui et al., 2006, Raghavendra et al., 2002, Song and Zhao, 2001, Tai et al., 2006) and simultaneously stimulate the production of proinflammatory cytokines (Johnston et al., 2004, Raghavendra et al., 2002, Raghavendra et al., 2003), both of which in turn play an active role in the development and maintenance of morphine tolerance. Moreover, that spinal neuroimmune responses are causal to, rather than simply associated with morphine tolerance, is further established by the finding that these responses are significantly abrogated by intrathecal injection of glial metabolism inhibitors and proinflammatory cytokine inhibitors (Raghavendra et al., 2002), or are accompanied with a down-regulation of glial GLAST and GLT-1 glutamate transporters (Tai et al., 2006). In sum, it is likely that opioid-induced/increased spinal neuroimmune activation undermines opioid-induced pain suppression (Watkins et al., 2007b).

However, most animal studies mentioned above were performed either in the absence of painful tissue injury which precluded extrapolation to the clinical situations (Zollner et al., 2008), or in neuropathic pain which demonstrated “naïve opioid tolerance” (Watkins et al., 2007b). Interestingly, some research claimed that opioid tolerance did not develop frequently in patients or animals with pathologic pain resulting from cancer (Portenoy and Lesage, 1999, Urch et al., 2005, Zech et al., 1995). Utilizing a rat model of inflammatory pain that mimics cancer associated pain, Zollner et al. (2008) indeed observed that chronic morphine application did not result in opioid tolerance, as the continuous availability of endogenous opioids increased recycling and preserved signaling of mu receptors in peripheral sensory neurons. As a unique pathological syndrome, CIBP possesses elements reminiscent of inflammatory, neuropathic, and cancer pain, but little is known about neuroimmune responses to chronic morphine administration in this particular pathological pain state. Thus, the aims of this study were to 1) investigate whether chronic systemic morphine treatment elicited pharmacological tolerance in a rat CIBP model induced by unilateral inoculation of Walker 256 mammary gland carcinoma cells into the tibia; and 2) examine the spinal neuroimmune activity after chronic morphine use in this model.

Section snippets

Radiological evaluation of bone destruction

All of the radiographs taken from carcinoma cells, chronic saline, and chronic morphine treated rats showed severe deterioration with full thickness unicortical bone loss on the postoperative day 19. This in combination with results from nociceptive behavioral tests suggested that the CIBP model was successfully established. No evident radiological difference was observed in the contralateral tibia bone from all of carcinoma cell inoculated rats (including carcinoma cell group, chronic saline

Discussion

The main findings of this study showed that prolonged morphine treatment failed to produce analgesic tolerance in this rat model of CIBP. Meanwhile, chronic morphine treatment had no significant influence on the spinal microglia and astrocytes according to their morphology, density, and expression of specific membrane surface markers, whereas it significantly down-regulated the production of spinal IL-1β and TNF-α at both mRNA and protein levels.

The underlying mechanisms of CIBP appear very

Preparation of animals

All experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and conducted in accordance with the NIH guide for the care and use of laboratory animals and Ethical Issue of the IASP (Zimmermann, 1983). Female Wistar rats, provided by the Institute of Laboratory Animal Science, Tongji Medical College, Huazhong University of Science and Technology, weighing 170–200 

Acknowledgments

The authors would like to thank Prof. De-Pei Li and Dr. Katherine Barker for proofreading this manuscript. This research was financially supported by the National Natural Science Foundation of China (no. 30672027, no. 30901396 and no. 30700783).

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    1

    These authors make equal contribution to this work.

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    Current address. Department of Anesthesiology and Pain Management MD Anderson Cancer Center, 1515 Holcombe Blvd, Houston, Texas 77030, USA.

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